The next generation of biosensing and biomedical devices requires cells to electrically interface with synthetic constructs, such as electrodes. However, cells are surrounded by insulating membranes that electrically isolate them from their environment. This proposal focuses on using directed evolution to engineer electron conduits that will allow recombinant cells to efficiently transport electrons across their membranes. A recent study has heterologously expressed three proteins, mtrA, mtrB, and mtrC, in Escherichia coli, allowing the recombinant microbe to transport electrons across its otherwise insulating membrane. Compared to natural systems that export electrons through the mtrABC pathway, the recombinant cells export electrons ten times slower. Recent findings suggest that inter-protein electron transfer in the recombinant cell is limiting. Consequently, we propose engineering the protein responsible for the bottleneck in the recombinant cell to improve the electron transfer efficiency. The specific aims are: (1) to develop a screen for assaying protein functionality in the electron transfer process, (2) to engineer the protein of interest to improve ts electron transfer efficiency via directed evolution, and (3) to evaluate whether extracellular electron flux is improved when the conduit is co-expressed with the evolved protein. Current data suggest that exogenous inter-protein interactions are limiting the electron flux, and this project proposes optimizing this unnatural interaction via directed evolution. These aims will be addressed using the tools and techniques available in the Frances Arnold group combined with assays developed by the Caroline Ajo-Franklin group in a close collaboration. The protein's ability to transfer electrons will be screened using a colorimetric assay that has been developed in the lab. This assay will be used to screen a library of mutant proteins. Residues responsible for modulating protein activity will be identified, elucidating the structure-function relation of he protein and helping understand the detailed mechanism of electron transport in this system. Finally, the relevant residues will be optimized, and the modified protein will be co-expressed with the conduit to evaluate improvements in extracellular electron transport. Extracellular transport will be monitored using both a colorimetric assay and electrode measurements.